Identification of CD8+ T cells that are CD161hi and/or IL18R(α)hi and have rapid drug efflux capacity

ABSTRACT

This invention provides, among other things, methods for the identification and isolation of viable putative long-lived antigen-specific memory CD8 +  T cell subsets (CMhi and EMhi) with high surface expression of CD161 and/or IL-18Rα and the capacity to rapidly efflux the fluorescent dye Rh123.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under AI053193 andCA114536 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 360056_423C1_SEQUENCE_LISTING.txt. The text fileis 2 KB, was created on Feb. 15, 2018, and is being submittedelectronically via EFS-Web.

BACKGROUND

Technical Field

The present invention concerns the utilization of high CD161 and/orIL-18Rα expression to identify putative long-lived rhodamine123-effluxing central (CM) and effector memory (EM) CD8⁺ T cells. Theinvention may have applications in adoptive T cell immunotherapy forcancer and infectious diseases, gene delivery, targeted ablationimmunotherapy and compositions useful therein.

Description of the Related Art

The ability to specifically recognize, control and eliminate infectionsand cancer is one of the hallmarks of human immunity. The immune systemcan be partitioned into 1) the non-specific ‘innate’ system, withresponses mediated by macrophages, dendritic cells, natural killer cellsand neutrophils and recognizing a relatively small number ofpathogen-associated molecular patterns, and 2) the highly specific CD4⁺and CD8⁺ T cell-mediated ‘adaptive’ immune system, potentiallyrecognizing millions of different peptide antigens.

Recognition of specific antigens by CD8⁺ T cells of the adaptive immunesystem is mediated by highly diverse T cell receptors (TCR). T cellsbearing a single TCR can recognize a specific peptide antigen presentedby an appropriate MHC molecule, resulting in an ‘adaptive’ immuneresponse with specificity for the presented peptide antigen. The CD8⁺ Tcell ‘adaptive’ immune response to ‘foreign’ antigens is wellcharacterized in viral infection, but CD8⁺ T cells specific for mutatedor non-mutated ‘self’ antigens may be found in other conditions such ascancer and autoimmune disease (1-7).

In acute viral infection, virus-derived antigens are processed andpresented by antigen presenting cells (APC) to naïve T cells thatexpress TCRs capable of recognizing the viral antigen and in humans arecharacterized by cell surface expression of CD45RA and CD62L and absenceof CD95 expression. The activated antigen-specific naïve T cells thenrapidly proliferate and differentiate into an effector T cellpopulation. The vast majority of effector T cells subsequently die, buta small fraction survives and become memory T cells (8, 9). Onre-challenge with the virus, the surviving memory T cell population hasthe capacity to rapidly proliferate and differentiate into an effectorpopulation to rapidly contain the infection and protect the host.Antigen-specific CD8⁺ T cell memory has been described to persist up to75 years, even in the absence of antigen rechallenge—essentiallyproviding immunity to that antigen for the lifetime of the host (10).

The surviving memory T cell population is highly heterogeneous andcomprises three main subsets in humans termed central memory (CM),effector memory (EM) and effector memory RA⁺ (EM_(RA)). These subsets,and sub-populations thereof, differ in phenotype, ontogeny, homing,proliferative capacity and cytokine secretion, and might have distinctroles in maintenance of immune memory (11-13). The distribution ofmemory subsets can be affected by variation in conditions at primingsuch as the nature of the APC and antigen, the antigen density and thepresence of cytokines, costimulatory molecules and inflammatorymediators (8). Once established, CD8⁺ T cell memory can persist in theabsence of antigen (14, 15). Memory CD8⁺ T cell populations undergohomeostatic (steady state) proliferation and different subsets appear tohave different rates of turnover in vivo (16). Interleukin-(IL-) 15 is acritical mediator of homeostatic proliferation and IL-7 is important forthe survival of established memory responses (17-22). Despite advancesin our understanding of the acute effector response to viral infectionand the transition to a stable memory response, the mechanisms by whichCD8⁺ memory is established and maintained have not been elucidated.

It has been hypothesized that a population of ‘stem-cell’ like T cellswith the capacity to self-renew and differentiate into effectors mayprovide for the maintenance of immunologic memory (23). A putativememory stem cell was recently identified in a murine model of graftversus host disease (GVHD) (24). After secondary transfer from mice withGVHD, only post-mitotic CD8⁺/CD44^(lo)/CD62L^(hi)/Sca-1^(hi) memorycells were able to initiate GVHD, give rise to memory (CM and EM) andeffector subsets, and retain replicative potential. Another study inmice demonstrated asymmetric cell division, a characteristic stem cellself-renewal mechanism, after the first encounter of naïve T cells withantigen (25). After the first division, the progeny ‘distal’ and‘proximal’ to the immunologic synapse were programmed for a memory andeffector phenotype, respectively. These studies suggest thatantigen-specific CD8⁺ T cell memory may be maintained by a long-livedpopulation with stem cell features and the capacity to self-renew. Todate, no candidate population has been identified in the human.

The identification of the phenotype of long-lived memory CD8⁺ T cells ormemory stem cells will have profound implications for investigation andtherapy of infections, cancer and autoimmune diseases. We have usedmultiparameter flow cytometry to identify memory CD8⁺ T cell populationsin humans with features consistent with stem cell behavior and longsurvival. A characteristic of hematopoietic and cancer stem cells is theability to efflux chemotherapy drugs and fluorescent dyes (26-30). Wefound that subpopulations of CM and EM CD8⁺ T cells also had thecapacity to rapidly efflux fluorescent dyes and chemotherapy drugs andwe hypothesized that these cells could be responsible for the observedchemoresistance of CD8⁺ T cell memory after severely myelosuppressivechemotherapy. In vitro studies demonstrated that CM and EM subsets withthe capacity to rapidly efflux rhodamine 123 (Rh123) (referred to asCMhi and EMhi, respectively for high efflux capacity) were moreresistant to apoptosis than their non-effluxing counterparts in responseto cytotoxic chemotherapy and that chemoresistance was attenuated byblockade of ATP-binding cassette cotransporter efflux channels.

Gene expression profiling studies show that CMhi and EMhi CD8⁺ T cellscomprise similar, yet distinct subsets. In addition, they have geneexpression profiles that are unique and distinct from those of othermemory or naïve CD8⁺ T cell populations. Further studies showed that theimmunophenotype of Rh123 effluxing memory populations was similar topreviously described ‘memory stem cells’ in mice and the ‘distalpole-derived memory cells’ after asymmetric division of naïve murineCD8⁺ T cells. CMhi and EMhi populations harbor CD8⁺ T cells expressing apolyclonal TCR repertoire and CMV, EBV and influenza antigen-specificCD8⁺ T cells can be identified within the subsets.

CMhi and EMhi CD8⁺ T cells are refractory to polyclonal stimulation withOKT3, demonstrating reduced proliferation and cytokine secretion,compared to non-effluxing CD8⁺ T cells. They also exhibit lowintracellular calcium flux in response to ionomycin stimulation. Thereduced proliferation and cytokine secretion can be partially rescuedwith costimulation and inflammatory cytokines. Despite the reduction insecretion of many inflammatory cytokines, CMhi and EMhi secreted IL-17in response to PMA-ionomycin stimulation in contrast to other memoryCD8⁺ T cell subsets.

The refractory nature of CMhi and EMhi may allow them to remain in aquiescent state, avoiding differentiation in response to antigenicstimulation in all but the most inflammatory conditions. Theobservations that CMhi show high ³H-thymidine uptake and CFSE dilutionin response to the homeostatic cytokines, IL-7 and IL-15, suggests thatthese chemoresistant cells may proliferate during the lymphocyte nadirafter myelosuppressive chemotherapy when IL-7 and IL-15 levels areelevated, and potentially repopulate the memory CD8⁺ T cell compartment.

CMhi and EMhi are found in very low numbers in cord blood. They arefound in high numbers in early adulthood and decline with advancing age.A population that arises after antigen exposure in early life and isgradually exhausted with repeated inflammatory antigenic stimuli inadulthood would be consistent with a putative memory stem cell. It wouldalso be consistent with recognized decreased efficacy of vaccination inelderly subjects.

Identification of CMhi and EMhi can be easily achieved in vitro usingRh123 efflux assays; however the use of Rh123 in functional studies orclinical grade isolation is problematic. Therefore, we usedmultiparameter flow cytometry to search for cell surface markers thatmight distinguish this subset of cells in human blood samples. We foundthat high expression of CD161 and/or IL-18Rα identifies subsets that areenriched in CMhi and EMhi, facilitating identification and isolation ofthese cells in the absence of in vitro culture or exposure of the cellsto Rh123 toxicity.

There is extensive evidence suggesting that memory CD8⁺ T cells have arole in prevention, control and therapy of infections, cancer andautoimmune diseases (1-7). Clinical studies of CD8⁺ T cell adoptivetransfer in stage IV melanoma resulted in up to 51% CR/PR anddemonstrated that persistence of the transferred tumor-specific T cellswas critical for efficacy (31, 32). Findings in CMV-specific adoptivetransfer studies also demonstrated the need for persistence, stronglysupporting the hypothesis that the establishment of long-lived memoryresponses may be essential for successful control and protection againsttumors and infection by adoptive T cell transfer (33). These studies arecomplemented by work in mice, demonstrating the critical role ofpersistent memory CD8⁺ T cells in eliminating clinically apparent cancerand establishing healthy equilibrium in occult cancer (4).

Despite the evidence that perturbations in memory CD8⁺ T cells areimportant in disease processes, attempts to specifically ablate, augmentor transfer antigen-specific immune memory have met with limitedsuccess. Clinical responses after transfer of CD8⁺ lines or clones havebeen shown previously, but have been sporadic, related to theirunpredictable persistence in vivo (32). Recent studies in non-humanprimates have shown CM-derived CD8⁺ clones can persist for up to oneyear after infusion, whereas EM-derived clones die rapidly by apoptosis,despite the fact that both populations shared an effector phenotypeprior to transfer (34). This suggests that effector CD8⁺ T cells mayretain an intrinsic program, derived from their cell of origin, whichdetermines their survival in vivo after antigenic stimulation and clonalexpansion. The implication is that clones or lines must be generatedfrom appropriately programmed subsets if transferred memory CD8⁺ T cellsare to persist in vivo. CMhi and EMhi are subsets with characteristicsthat suggest they are programmed for long survival.

The identification of memory CD8⁺ T cell subsets with appropriateprogramming for persistence will facilitate transfer of CD8⁺ Tcell-mediated immunity against specific antigens. CD8⁺ T cells withprogramming for persistence and long-lived survival may also be usefulas delivery vehicles for therapeutic genes. In addition to beingamenable to long-term survival for maintenance of therapeutic immunityor gene delivery after adoptive transfer, CMhi and EMhi long-livedmemory cells may act as targets for immunosuppression by ablation ofantigen-specific memory responses through the use of toxin-conjugatedCD161 and/or IL-18Rα monoclonal antibodies. This form of therapy mayhave a role in autoimmune diseases and graft versus host disease.

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BRIEF SUMMARY

Identification of long-lived memory cells in humans has not beenpossible to date. We demonstrate that high CD161 and/or IL-18Rαexpression may be used to identify human Rh123 effluxing long-livedmemory cells in the CM and/or EM CD8⁺ T cell compartments. Thesepopulations may be useful as a source of T cells for adoptiveimmunotherapy, gene delivery or as targets for ablation in autoimmune orother immunopathologic conditions.

This invention provides methods for the identification and isolation ofviable putative long-lived antigen-specific memory CD8⁺ T cell subsets(CMhi and EMhi) with high surface expression of CD161 and/or IL-18Rα andthe capacity to rapidly efflux the fluorescent dye Rh123.

We propose that CMhi and EMhi cells are programmed for long in vivosurvival and chemoresistance, consistent with their sharedcharacteristics with stem cell populations in other tissues. Theidentification of a distinguishing phenotype, including the high surfaceexpression of CD161 and/or IL-18Rα will enable the isolation and invitro manipulation of these T cell subsets. The ability to isolate andadoptively transfer long-lived T cells in an autologous, allogeneic orsyngeneic setting may enable transfer of persistent antigen-specificimmunity. This may be important in a range of applications, including,but not limited to prevention, control or elimination of cancer orinfection. Alternatively, targeted ablation of CMhi and EMhi populationsmay result in potent immunosuppression and treatment of autoimmunediseases or graft versus host disease. Candidate immunomodulatorytherapies, such as vaccines or immunosuppressant drugs, may be screenedin high throughput in vitro assays using the invention. The inventionmay also be used as an in vivo delivery vehicle for therapeutic genes.

Stated otherwise, a first aspect of the invention is a human CD8⁺ Tlymphocyte that has high expression of CD161 and/or IL-18Rα (e.g., alymphocyte that actively effluxes Rh123 in less than 30 minutes culturein RPMI 1640/10% BSA at 37° C. (a “rapidly effluxing and/or CD161^(hi)and/or IL-18Rα^(hi) T lymphocyte”). The lymphocytes may be provided inisolated, enriched and/or purified form.

In some embodiments, the lymphocyte is TCRαβ⁺, TCRγδ⁻, CD8α^(hi/int),CD8β^(hi/int/neg), CD45RA^(int/neg), CD45RO^(int/hi), CD95^(int/hi),CD25^(int/neg), CD27⁺, CD28⁺, CD57⁻, CD127⁺ CD103⁻, PD-1^(neg),bcl-2^(hi), bcl-xL^(hi), perforin^(int), granzyme A^(int), granzymeB^(int/neg) and NKG2D^(int).

In some embodiments, the lymphocyte has high expression of MDR-1 mRNA.

In some embodiments, the lymphocyte has the capacity to actively effluxthe fluorescent chemotherapy drug, daunorubicin.

In some embodiments, the lymphocyte is resistant to apoptosis afterculture with 0.03-0.3 μM daunorubicin and such resistance is abrogatedby the efflux inhibitor PK11195.

In some embodiments, the lymphocyte demonstrates absent or lowerproliferation in response to stimulation with 250-1000 ng/ml plate-boundOKT3 than its non-effluxing counterpart T lymphocyte. The reducedproliferation can be partially recovered by costimulation withplate-bound anti-CD28 antibody and/or addition of cytokines including,but not limited to, IL-7, IL-12, IL-15, IL-18 and IL-23 or combinationsthereof.

In some embodiments, the lymphocyte has reduced secretion of IFN-γ,IL-2, IL-4, IL-5, IL-8, IL-10 and MIP-1α compared to their non-effluxingcounterparts in response to stimulation with PMA/ionomycin or OKT3 incombination with anti-CD28 antibody or cytokines.

In some embodiments, the lymphocyte has low, but heterogeneous, calciumflux after stimulation with ionomycin;

In some embodiments, the lymphocyte is a CD62L⁺ central memory T cell.

In some embodiments, the lymphocyte demonstrates high uptake of³H-thymidine in response to 4 days culture with 0.5-2.0 ng/ml IL-7 andgreater dilution of CF SE in response to 8-11 days culture with 0.5-2.0ng/ml IL-7, as compared to its non-effluxing counterpart T lymphocyte.

In some embodiments, the lymphocyte is a CD62L⁻ effector memory T cell.

A further aspect of the invention is lymphocytes as described herein foruse in adoptive immunotherapy or gene therapy, or for use in thepreparation of a medicament for adoptive immunotherapy or gene therapy.

A further aspect of the invention is a composition comprising,consisting of or consisting essentially of lymphocytes as describedherein in a pharmaceutically acceptable carrier.

A further aspect of the invention is a method of treating a humansubject in need thereof, comprising administering lymphocytes asdescribed herein to the said subject in a treatment-effective amount.The lymphocytes may be autologous, allogeneic, or syngeneic cells. Themethod may be carried out for immunotherapy or adoptive immunotherapy;the method may be carried out where the subject is afflicted with atleast one of: cancer, infectious disease, or iatrogenic or congenitalimmunodeficiency; the method may be carried out for gene therapy; themethod may be carried out where the subject is afflicted with one of:congenital genetic disorder, cancer, infectious disease, or iatrogenicor congenital immunodeficiency. A typical example of the use of theinvention in adoptive immunotherapy would be treatment in the setting ofallogeneic or autologous hematopoietic stem cell transplantation (HSCT).In the setting of severe immunosuppression, allogeneic and autologousHSCT patients are at great risk of contracting opportunistic infections.The invention may be used to transfer immune memory to the recipient toprotect them against potentially life-threatening infection. CMhi andEMhi cells may be isolated from the transplant donor (in the case ofallogeneic HSCT) or the patient (in the case of autologous HSCT),selected or engineered for specificity to infection-associated antigens,expanded or not, and returned to the patient in the post transplantsetting. The subsets would proliferate with or without differentiationin the post-transplant lymphopenic environment and reconstitute immunememory. The selection of CMhi and EMhi specific for infections wouldallow reconstitution of immunity without causing graft versus hostdisease.

A further aspect of the invention is a method of treating a humansubject in need thereof, comprising ablating rapidly effluxing and/orCD161 and/or IL-18Rα^(hi) T lymphocytes in said subject by atreatment-effective amount. The ablating step can be carried out in vivoor in vitro. In some embodiments, the subject is afflicted with or atrisk for at least one of: of autoimmune disease, graft versus hostdisease or rejection of a transplant graft. In some embodiments theablating step is carried out in vivo by direct treatment of the patient;in some embodiments the ablating step is carried out ex vivo bydepletion of cells from a cellular product or extracorporealcirculation; in some embodiments the ablating step is carried out byablating said cells from a transplant graft prior to transplantation. Insome embodiments the ablating step is carried out by administeringanti-CD161 and/or anti-IL-18Rα monoclonal or polyclonal antibodies,conjugated or not to toxic groups or radioisotopes, lymphotoxic drugtreatment with inhibition of efflux pumps, or combinations thereof.

A further aspect of the invention is a kit for the identification orisolation of rapidly effluxing T lymphocytes as described herein,comprising a combination of at least two, three or four, in anycombination, of: (a) a combination of fluorochrome-conjugated antibodiesto allow identification of central and effector memory CD8⁺ T cells; (b)a fluorochrome-conjugated antibody to CD161 and/or IL-18Rα andappropriate isotype control; (c) Rh123 and a negative control antagonistof Rh123 efflux; (d) appropriate assay medium; and (e) instructions andpackaging.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: CM and EM populations contain subsets that rapidly efflux Rh123and are CD161^(hi) and IL-18Rα^(hi) as described in Example 1.

FIGS. 2A-2C: CMhi and EMhi subsets are TCRαβ⁺ and TCRγδ⁻. There is norestriction to Vα24. They are predominantly CD45RA^(int/neg),CD45RO^(int/hi), CD95⁺, CD8α⁺, CD8β^(+/neg), CD27⁺, CD28⁺, CD122⁺,perforin^(int/lo), granzyme A^(int/lo), granzyme B^(neg), Ki67^(neg),bcl-xL^(int/hi) and bcl-2^(int/hi), as described in Example 2.

FIG. 3: CMhi and EMhi have a surface phenotype that suggests possiblederivation from the distal pole of an asymmetrically dividing naïve CD8⁺T cell, as described in Example 3.

FIGS. 4A-4B: CMhi and EMhi express higher levels of MDR-1 mRNA thantheir non-effluxing counterparts and actively efflux the fluorescentchemotherapy drug, daunorubicin, as described in Example 4.

FIG. 5: CMhi and EMhi are resistant to daunorubicin-induced apoptosis invitro, as described in Example 5.

FIGS. 6A-6D: CMhi and EMhi divide in response to the homeostaticcytokines, IL-7 and IL-15, and have high viability after culture in theabsence of supplementary cytokines, as described in Example 6.

FIG. 7: CMhi and EMhi show reduced ³H-thymidine uptake in response topolyclonal TCR stimulation with OKT3, compared to their non-effluxingcounterparts, as described in Example 7. ³H-thymidine uptake isincreased after costimulation as indicated in the Figure.

FIG. 8: CMhi and EMhi have a different cytokine secretion profilecompared to their non-effluxing counterparts, as described in Example 8.

FIG. 9: CMhi and EMhi have decreased calcium flux in response toionomycin, compared to their non-effluxing counterparts, as described inExample 9.

FIG. 10: CMhi and EMhi subsets comprise polyclonal TCR repertoires bymolecular spectratyping, as described in Example 10.

FIGS. 11A-11B: Viral antigen tetramer-positive cells can be identifiedwithin CMhi and EMhi subsets and CMV-, EBV- and influenza-specific CTLresponses can be generated from sorted CMhi and EMhi subsets, asdescribed in Example 11.

FIG. 12: CMhi and EMhi subsets have unique and distinct gene expressionprofiles as shown by the Principal Components Plot and as described inExample 12.

FIGS. 13A-13B: CMhi and EMhi are rare in cord blood, peak in early adultlife and are found at decreasing frequency with advancing age, asdescribed in Example 13.

The present invention is explained in greater detail below. Thedisclosures of all United States patent references cited herein areincorporated by reference herein in their entirety.

DETAILED DESCRIPTION

I. Definitions

“T cells” or “T lymphocytes” as used herein are from humans. In someembodiments the T cells are autologous (the donor and the recipient arethe same individual); in some embodiments the T cells are allogeneic(the donor and recipient/s are genetically different individuals); insome embodiments the T cells are syngeneic (the donor and recipient/sare different individuals who are genetically identical).

“Cytotoxic T lymphocyte” (CTL) as used herein refers to a T lymphocytethat expresses CD8 on the surface thereof (i.e., a CD8⁺ T cell).

“Central memory” T cell (or “CM”) as used herein refers to a CTL thathas previously been exposed to antigen (a “memory CTL”) and is CD62L⁺,CD45RA^(int/neg)/CD45RO^(int/hi) and CD95^(int/hi).

“Effector memory” T cell (or “EM”) as used herein refers to a CTL thathas previously been exposed to antigen (a “memory CTL”) and is CD62L⁻,CD45RA^(int/neg)/CD45RO^(int/hi) and CD95^(int/hi).

“Rapidly effluxing T lymphocytes” are found within the CD8⁺ T cell CMand EM populations (CMhi and EMhi, respectively) with some or all of thefollowing identifying characteristics: (a) actively and rapidly effluxthe dye Rh123 in culture over a time of 30 minutes at a temperature of37° C.; (b) high surface expression of CD161 (CD161^(hi)) and/or IL-18Rα(IL-18Rα^(hi)). Such cells typically include, but are not limited to,the following features: (a) high surface expression of CD127, CD28; (b)typically, but not consistently, expression of markers suggestingderivation from the distal pole complex or uropod of mitotic CD8⁺ Tcells (higher CD43, CD44, CD46, CD148 and CD162 surface expression thannon-effluxing subsets; lower CD8, CD11 a and CD50 surface expressionthan non-effluxing subsets); (c) CD3⁺/TCRαβ⁺/TCRγδ⁻; (d) low or nosurface expression of CD25, CD57, PD-1, CD103 and CD69; (e) lowersurface expression of NKG2D than non-effluxing subsets; (f)intermediate/high expression of CD45RO, intermediate/negative surfaceexpression of CD45RA and expression of CD95; (g) typically, but notconsistently higher expression of bcl-2 and bcl-xL than theirnon-effluxing counterparts; (h) negative expression of granzyme B,intermediate expression of granzyme A and low/intermediate expression ofperforin; (i) normal or low expression of CD8α and normal, low or absentexpression of CD8β; (j) higher expression of MDR-1 mRNA than theirnon-effluxing counterparts; (k) lower percentage expression of Ki67 thantheir non-effluxing counterparts in a healthy individual. Optionally, insome embodiments, rapidly effluxing T lymphocytes are characterized bypositive surface expression of CD122. Note also that CD62L expression isvariable and defines the presence of these cells in the CM or EMsubsets. Such cells also typically include, but are not limited to, thefollowing functional features: (a) low proliferation compared tonon-effluxing subsets in response to stimulation with plate-bound OKT3,which can be increased after costimulation with anti-CD28 antibody orcytokines; (b) contain virus-specific clones, evidenced by tetramerbinding and the capacity to identify antigen-specific CD8⁺ T cells afterin vitro expansion of sorted subsets; (c) resistance to apoptosis duringin vitro culture in the presence or absence of chemotherapeutic agents;(d) reduced secretion of IFN-γ, IL-2, IL-4, IL-5, IL-8, IL-10 and MIP-1αcompared to their non-effluxing counterparts in response to stimulationwith PMA/ionomycin or OKT3 in combination with anti-CD28 antibody orcytokines; (e) capacity to actively efflux the fluorescent chemotherapydrug, daunorubicin; (f) low, but heterogeneous, calcium flux afterstimulation with ionomycin; (g) CMhi cells typically have an increaseduptake of tritiated thymidine (³H-thymidine) and increased dilution ofCFSE compared to their non-effluxing counterparts in response to IL-7.

“Efflux blockers” as used herein includes, but is not limited to,PK11195, cyclosporine A, PSC833, verapamil.

“Effluxed drug” as used herein includes, but is not limited to,doxorubicin, daunorubicin, epirubicin, methotrexate, mitoxantrone,vinblastine, vincristine, dexamethasone and derivatives, etoposide andtaxanes.

“Antigen” as used herein refers to a protein or peptide that can berecognized by the immune system.

“Antigen-specific” as used herein refers to the nature of the highlyspecific recognition by the adaptive immune system of peptide fragmentspresented in the context of an MHC molecule.

“Antibody” as used herein refers to all types of immunoglobulins,including IgG, IgM, IgA, IgD, and IgE and all isotypes thereof. Of theimmunoglobulins, IgM and IgG are particularly preferred. The antibodiesmay be monoclonal or polyclonal and may be of any species of origin,including (for example) mouse, rat, rabbit, horse, or human, or may bechimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26,403-11 (1989). The antibodies may be recombinant monoclonal antibodiesproduced according to the methods disclosed in Reading U.S. Pat. No.4,474,893, or Cabilly et al., U.S. Pat. No. 4,816,567. The antibodiesmay also be chemically constructed by specific antibodies made accordingto the method disclosed in SegAl et al., U.S. Pat. No. 4,676,980.

“Autoimmune disease” as used herein may be any autoimmune disease,including but not limited to: systemic lupus erythematosus, Hashimoto'sdisease, rheumatoid arthritis, graft-versus-host disease, Sjogren'ssyndrome, pernicious anemia, Addison's disease, scleroderma,Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia,sterility, myasthenia gravis, multiple sclerosis, Basedow's disease,thrombotic thrombocytopenic purpura, immune thrombocytopenic purpura,insulin-dependent diabetes mellitis, allergy, asthma, atopic disease,arteriosclerosis, myocarditis, cardiomyopathy, nephritis, andhypoplastic anemia. See, e.g., U.S. Pat. No. 7,279,160.

“Cancer” as used herein may include, but is not limited to anypathologic variation of: cancer of the prostate, breast, bladder,stomach, oropharynx, nasopharynx, esophagus, stomach, pancreas, liver,kidneys, colon, rectum, anus, lung, thyroid, brain, hematopoietic system(including, but not limited to Hodgkin's and Non-Hodgkin's lymphoma,acute and chronic lymphoid and myeloid leukemias) and skin (including,but not limited to, basal cell carcinoma, squamous cell carcinoma andmelanoma).

“Donor” as used herein refers to the individual from whom rapidlyeffluxing T lymphocytes or other cellular product was obtained.

“Recipient” as used herein refers to the individual who will receiverapidly effluxing T lymphocytes or other treatment.

“Enriched” as used herein to describe amounts of cell types in a mixturerefers to the subjecting of the mixture of the cells to a physicalprocess or step, which results in an increase in the number of the“enriched” type, as compared to the same mixture before that physicalprocess or step. For cell types that are few in number, those cells maybe enriched five, ten, twenty, thirty, forty, or fifty fold (times) ormore, yet still be a relatively small number of total cells (e.g., totalnumber T cells) in the “enriched” population (e.g., the enriched cellsbeing at least 1, 5, 10, 20, or 30 percent of the total cells in thepreparation, or more, up to 40 or 50 percent of the total cells in thepreparation or more). In other embodiments, the “enriched” cells may beenriched to a point that they become at least 40, 50, 60, 70 or 80percent of the total cells in the preparation, or more, up to 90, 95, or99 percent of the total cells in the preparation, or more).

“Toxic agent” as used herein includes, but is not limited to,radioisotopes, therapeutic drugs, and toxins or cytotoxins. See, e.g.,U.S. Pat. No. 6,274,118.

“Radioisotope” as used herein includes but is not limited to. ²²⁷Ac,²¹¹At, ¹³¹Ba, ⁷⁷Br, ¹⁰⁹Cd, ⁵¹Cr, ⁶⁷Cu, ¹⁶⁵Dy, ¹⁵⁵Eu, ¹⁵³Gd, ¹⁹⁸Au,¹⁶⁶Ho, ^(113m)In, ^(115m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁹Ir, ¹⁹¹Ir, ¹⁹²Ir,¹⁹⁴Ir, ⁵²Fe, ⁵⁵Fe, ⁵⁹Fe, ¹⁷⁷Lu, ¹⁰⁹Pd, ³²P, ²²⁶Ra, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm,⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ⁷⁵Se, ¹⁰⁵Ag, ⁸⁹Sr, ³⁵S, ¹⁷⁷Ta, ^(117m)Sn, ¹²¹Sn,¹⁶⁶Yb, ¹⁶⁹Yb, ⁹⁰Y, ²¹²Bi, ¹¹⁹Sb, ¹⁹⁷Hg, ⁹⁷Ru, ¹⁰⁰Pd, ^(101m)Rh, and²¹²Pb.

“Therapeutic drug” as used herein includes but is not limited toAdriamycin, Chlororambucil, Daunorubicin, Leucovorin, Folinic acid,Methotrexate, Mitomycin C, Neocarzinostatin, Melphalan Vinblastine,Mitocyn, Mechlorethamine, Fluorouracil, Floxuridine, Idarubicin,Doxorubicin, Epirubicin, Cisplatin, Cannustine, Cyclophosphamide,Bleomycin, Vincristine and Cytarabine.

“Toxin” or “cytotoxin” as used herein includes but is not limited todiptheria toxin, ricin toxin, monensin, verrucarin A, abrin, saporin,vinca alkaloids, tricothecenes, and pseudomonas exotoxin A, and pokeweedviral protein. See, e.g., U.S. Pat. No. 6,630,576.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

II. Identification

Identification of Rh123-Effluxing CM and EM Populations. PBMC or Tlymphocytes from any tissue can be collected in accordance with knowntechniques and loaded with Rh123 (or alternate similarly-effluxedfluorescent dye) at 5-10 μg/ml in RPMI 1640/10% BSA (efflux buffer) onice for 30 minutes, washed three times and cultured in efflux buffer for30 minutes at 37° C. A control sample cultured in the presence ofvinblastine (a competitive antagonist of Rh123 efflux) can be used toestablish and gate active efflux of Rh123. The PBMC can then be surfacelabeled with fluorochrome-conjugated antibodies (e.g., to CD4, CD16,TCRγδ, Vα24, CD8α, CD95 and CD62L), allowing identification ofRh123^(lo) effluxing CM or EM populations by fluorescence-activated cellsorting (FACS) analysis.

Identification of CD161^(hi) and/or IL-18Rα^(hi) CM and EM Populations.High expression of CD161 and/or IL-18Rα on CM and EM populations can beused as a surrogate marker for the ability to rapidly efflux Rh123 in invitro culture. T lymphocytes can be collected in accordance with knowntechniques from any tissue and labeled with fluorochrome-conjugatedantibodies to CD161 and/or IL-18Rα, and other markers (e.g., to CD4,CD16, TCRγδ, Vα24, CD8α, CD95 and CD62L), allowing identification ofCD161^(hi) and/or IL-18Rα^(hi) effluxing CM (CMhi) or EM (EMhi)populations by fluorescence-activated cell sorting (FACS) analysis.

III. Isolation

Isolation of Rh123-Effluxing CM and EM Populations. PBMC or Tlymphocytes from any tissue can be collected in accordance with knowntechniques and enriched or depleted by known techniques such as affinitybinding to antibodies such as in flow cytometry, immunomagneticseparation and/or affinity binding. For example, CD8⁺ CTL may beisolated by positive immunomagnetic separation. The positively selectedCD8⁺ T cell fraction can be loaded with Rh123 (or alternatesimilarly-effluxed fluorescent dye) at 5-10 μg/ml in efflux buffer onice for 30 minutes, washed three times and cultured in efflux buffer for30 minutes at 37° C. A control sample cultured in the presence ofvinblastine (a competitive antagonist of Rh123 efflux) can be used toestablish and gate active efflux of Rh123. The PBMC can then be labeledwith antibodies (e.g., to CD4, CD16, TCRγδ, Vα24, CD8α, CD95 and CD62L),allowing identification and isolation ofCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8α⁺/CD95⁺/CD62L⁺/Rh123^(lo) effluxing CMhi orCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8α⁺/CD95⁺/CD62L⁻/Rh123^(lo) effluxing EMhipopulations by fluorescence-activated cell sorting (FACS) analysis.

Isolation of CD161^(hi) and/or IL-18Rα^(hi) CM and EM Populations. Highexpression of CD161 and/or IL-18Rα on CM and EM populations can be usedas a surrogate marker for the ability to rapidly efflux Rh123 in invitro culture. T lymphocytes from any tissue can be collected inaccordance with known techniques and enriched or depleted by knowntechniques such as affinity binding to antibodies such as in flowcytometry, immunomagnetic separation and/or affinity binding. Forexample, CD8⁺ CTL may be isolated by positive immunomagnetic separation.The positively selected CD8⁺ T cell fraction can be labeled withantibodies (e.g., to CD4, CD16, TCRγδ, Vα24, CD8α, CD95, CD62L and CD161and/or IL-18Rα), allowing identification and isolation ofCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8α⁺/CD95⁺/CD62L⁺/CD161^(hi) orCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8α⁺/CD95⁺/CD62L⁺/IL-18Rα^(hi) effluxing CMhior CD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8α⁺/CD95⁺/CD62L⁻/CD161^(hi) orCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8α⁺/CD95⁺/CD62L⁻/IL- 18Rα^(hi) effluxing EMhipopulations by fluorescence-activated cell sorting (FACS) analysis.

IV. Kits. Kits useful for carrying out all or parts of the methods ofthe invention (particularly the steps of identifying or isolating cellpopulations) may take any of a variety of forms. Typically the kitswould include the necessary antibodies or antibody conjugates for theprocedure. Isolation of these cells is a multistep process. Manycombinations of kits are possible, depending on the desired product(pure CD8⁺ CMhi cells, pure CD8⁺ EMhi cells or enriched CD8⁺ CMhi andEMhi cells) and method of identification of CMhi and EMhi (Rh123effluxing and/or CD161^(hi) and/or IL-18Rα^(hi)). The kit may be usedfor identification and analysis, non-clinical grade or clinical gradeisolation of Rh123 effluxing or non-effluxing populations. The kit canbe packaged in any suitable container and optionally includeinstructions for carrying out all or parts of the methods describedherein. Hence, a kit could include, but is not limited to, anycomponents necessary for any combination of the following steps:

(i) Initial identification, enrichment and/or isolation of memory CD8⁺ Tcells (positive or negative immunomagnetic selection or cell sorting)

(ii) Removal of contaminating cells after immunomagnetic separation(e.g., antibodies to non-CD8⁺ T cells, streptavidin-fluorochromeconjugates, goat anti-mouse-fluorochrome conjugates).

(iii) Identification, enrichment and/or isolation of CM and EM subsets(e.g., fluorochrome-conjugated CD95 and CD62L)

(iv) Identification, enrichment and/or isolation of effluxing(Rh123/efflux buffer/efflux blocking agents) or CD161^(hi) and/orIL-18Rα^(hi) populations

V. Compositions and Methods for Therapy

CMhi and EMhi cells may be used in an autologous, allogeneic orsyngeneic setting. CMhi and EMhi populations may be used or targetedfor, but are not limited to, the following indications;

(a) for transfer of antigen-specific immunity in cancer, infectiousdiseases or immunodeficiency

(b) for targeted ablation of immunity in autoimmune diseases, graftversus host disease or graft rejection

(c) for therapeutic gene delivery

VI. Transfer of Antigen-Specific Immunity

In some embodiments, the lymphocytes of the invention may be used toconfer immunity to individuals. By “immunity” is meant an increase ofone or more factors associated with a response to infection by apathogen, or to a tumor, to which the lymphocyte response is directed.

Subjects that can be treated by the present invention are human. Thesubjects can be male or female and can be any suitable age, includinginfant, juvenile, adolescent, adult, and geriatric subjects.

Subjects that can be treated include subjects afflicted with cancer,including but not limited to hematopoietic cancers, cancers of thecolon, lung, liver, breast, prostate, ovary, skin (including melanoma),bone, and brain etc. In some embodiments the tumor associated antigensare known, including, but not limited to melanoma, breast cancer,squamous cell carcinoma, colon cancer, leukemia, myeloma and prostatecancer (in these embodiments memory T cells can be isolated orengineered by introducing the T cell receptor genes). In otherembodiments the tumor associated antigens can be targeted withgenetically modified T cells expressing an engineered immunoreceptor.Examples include but are not limited to B cell lymphoma, breast cancer,prostate cancer, and leukemia.

Subjects that can be treated also include subjects afflicted with, or atrisk of developing, an infectious disease, including but not limited toviral, bacterial, and protozoal infections.

Subjects that can be treated include immunodeficient patients, includingbut not limited to transplant patients, afflicted with a viralinfection. Such viral infections may include, but are not limited to,cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, influenzaor parainfluenza, varicella, human herpes virus type 6 (HHV6) or HHV7,respiratory syncytial virus (RSV) or BK polyomavirus infections.

Cells prepared as described above can be utilized in methods andcompositions for adoptive immunotherapy in accordance with knowntechniques, or variations thereof that will be apparent to those skilledin the art based on the instant disclosure (see, e.g., US PatentApplication Publication No. 2003/0170238 to Gruenberg et al; see alsoU.S. Pat. No. 4,690,915 to Rosenberg; see also U.S. Pat. No. 6,040,177to Riddell).

CMhi and EMhi may be used for therapy of autologous, allogeneic orsyngeneic recipients and may be harvested in the presence or absence ofin vivo stimulation, and administered with or without in vitromanipulation or in vivo stimulation after administration.

In vivo stimulation before harvesting CMhi and/or EMhi may involve, butis not limited to, the use of drugs, vaccines or cytokines. In vitromanipulation may involve, but is not limited to, by culture or othermethods, any combination of subset enrichment, antigen-specificenrichment, expansion, feeder cell (e.g., irradiated LCL or PBMC)treatment, cytokine treatment, antibody treatment, small moleculetreatment or transduction with antigen-specific TCR genes or othertherapeutic, suicide and/or knockdown genes. In vivo stimulation mayinvolve, but is not limited to, vaccination with commercial vaccines orresearch cellular or non-cellular reagents, cytokine administration ordrug administration.

CMhi or EMhi cells may be infused with or without isolation andenrichment. The use and method of enrichment or isolation will reflectthe requirements of the product. In some embodiments, the cells areformulated by first harvesting them from the donor or their culturemedium, and then washing and concentrating the cells in a medium andcontainer system suitable for administration (a “pharmaceuticallyacceptable” carrier) in a treatment-effective amount. Suitable infusionmedium can be any isotonic medium formulation, such as normal saline,Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water orRinger's lactate. The infusion medium can be supplemented with humanserum albumin, human serum or other nutrients.

The amount of cells to be infused is variable and may range from <10antigen-specific cells in the absence of enrichment or in vitroexpansion to greater than 10¹² cells after enrichment and in vitroexpansion (see, e.g., U.S. Pat. No. 6,040,177 to Riddell et al. atcolumn 17). The cells may be administered by a single infusion or bymultiple infusions over a range of time. However, since differentindividuals are expected to vary in responsiveness, the type and amountof cells infused, the route of administration, the number of infusionsand the time range over which multiple infusions are given aredetermined by the attending physician as dictated by circumstance,physical and laboratory examination.

A typical example of the use of the invention in adoptive immunotherapywould be treatment in the setting of allogeneic or autologoushematopoietic stem cell transplantation (HSCT). In the setting of severeimmunosuppression, allogeneic and autologous HSCT patients are at greatrisk of contracting opportunistic infections. The invention may be usedto transfer immune memory to the recipient to protect them againstpotentially life-threatening infection. CMhi and EMhi cells may beisolated from the transplant donor (in the case of allogeneic HSCT) orthe patient (in the case of autologous HSCT), selected or engineered forspecificity to infection-associated antigens, expanded or not, andreturned to the patient in the post transplant setting. The subsetswould proliferate with or without differentiation in the post-transplantlymphopenic environment and reconstitute immune memory. The selection ofCMhi and EMhi specific for infections would allow reconstitution ofimmunity without causing graft versus host disease.

VII. Targeted Ablation

A further aspect of the invention is a method of ablating long-livedmemory CD8⁺ T cells in a human subject by selectively depleting activelyeffluxing CMhi and/or EMhi cells in the subject as treatment forautoimmune disease or graft versus host disease. The selective depletionmay be carried out by any suitable technique, such as by administeringto the subject an antibody that selectively binds to CD161 and/orIL-18Rα in an amount effective to treat the disease or by administeringinhibitors of drug efflux in combination with cytotoxic drugs.

The antibody, either monoclonal or polyclonal of any isotype, orpharmaceutical composition containing the same, can be formulated inmultiple different carriers, or conjugated to many possible toxins,including but not limited to radioisotopes, ricin or diphtheria toxin,as is known in the art. The antibody could be administered by variousroutes including but not limited to, intravenously, intraperitoneally orintracisternally. A therapeutic antibody of the invention isadministered in an effective amount to treat the disease, at a suitableschedule, though these will vary somewhat with the particular disease,formulation, route of administration, and condition of the subject, asis known in the art.

CMhi and EMhi may be ablated by administration of cytotoxic drugs incombination with inhibitors of cytotoxic drug efflux. In vitro studiesshow that CMhi and EMhi are protected from chemotherapy-inducedapoptosis and this protection is lost in the presence ofABC-cotransporter (drug efflux pump) inhibition. Prevention of cytotoxicdrug efflux from CMhi and EMhi may be used to reduce or ablate thesecell populations in vivo. Cytotoxic drugs and drug efflux inhibitors maybe administered in recognized or novel protocols as is known in the art.

VIII. Therapeutic Gene Delivery

In some embodiments, the lymphocytes of the invention may be used todeliver therapeutic genes to individuals. Therapeutic genes couldinclude, but are not limited to, genes encoding costimulatory (e.g.,CD80) or inhibitory molecules (PD-L1), cytokines (e.g., IL-7),apoptosis-inducing signals or congenitally deficient or abnormal genes(e.g., Factor VIII gene in severe hemophilia). The delivery of T cellstransfected with genes encoding antigen-specific TCR is discussed abovein “Transfer of antigen-specific immunity”.

Isolation, in vitro manipulation, formulation and administration fortherapeutic gene delivery will encompass similar considerations, asdiscussed above in “Transfer of antigen-specific immunity”. Genedelivery to isolated T cells would likely be performed in vitro, but mayinclude methods to target long-lived memory cells in vivo. Utilizingrecombinant infectious virus particles for gene delivery is a preferredapproach to the transduction of T lymphocytes of the present invention.The viral vectors which have been used in this way include virus vectorsderived from simian virus 40, adenoviruses, adeno-associated virus(AAV), retroviruses and lentiviruses and modifications thereof. Thus,gene transfer and expression methods are numerous but essentiallyfunction to introduce and express genetic material in mammalian cells.Several of the above techniques, amongst others, have been used totransduce hematopoietic or lymphoid cells, including calcium phosphatetransfection, protoplast fusion, electroporation, and infection withrecombinant adenovirus, adeno-associated virus and retrovirus vectors.Primary T lymphocytes have been successfully transduced byelectroporation, retroviral infection and lentiviral infection.

EXPERIMENTAL Example 1 CM and EM Populations Contain Subsets ThatRapidly Efflux Rh123 and are CD161^(hi) and IL-18Rα^(hi)

PBMC were separated from fresh peripheral blood by density gradientcentrifugation, washed in RPMI 1640/10% bovine serum albumin (hereinknown as efflux buffer) and resuspended at 1×10⁶/ml in ice cold effluxbuffer with 10 μg/ml Rh123. PBMC were incubated for 30 minutes on icebefore washing three times in ice cold efflux buffer and resuspending inpre-warmed efflux buffer, with or without vinblastine, for 30 minutes at37° C. At 30 minutes, PBMC were washed once in ice cold PBS/0.2% BSA(FACS buffer) and labeled with antibodies to CD4, CD16, TCRγδ, Vα24,CD3, CD8α, CD95, CD62L, CD161 and IL-18Rα for 20 minutes on ice. Afterwashing in ice cold FACS buffer, samples were analyzed on a BD FacsARIAflow cytometer.

CM and EM subsets were identified as CD62L⁺ or CD62L⁻ events,respectively, in the CD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD3⁺/CD8⁺/CD95⁺ population.Rh123 fluorescence was identified with appropriate compensation and a530/30 emission filter after laser excitation at 488 nm. Rh123-effluxingevents were defined as those with fluorescence lower than the meanfluorescence intensity identified after culture in the presence ofvinblastine efflux blockade.

The results in FIG. 1 demonstrate that many lymphocytes have thecapacity to efflux Rh123; however only small subsets of CM and EM CD8⁺ Tlymphocytes have the capacity to rapidly efflux Rh123 over a 30 minuteperiod. Efflux is blocked by vinblastine, a non-fluorescent substratefor MDR-1 and MRP-1, demonstrating specificity of Rh123 efflux. CM andEM cells rapidly effluxing Rh123 express high levels of IL-18Rα andCD161.

Example 2 CMhi and EMhi Subsets are TCRαβ⁺ and TCRγδ⁻. There is NoRestriction to Vα24. They are Predominantly CD45RA^(int/neg),CD45RO^(int/hi), CD95⁺, CD8α⁺, CD8β^(+/neg), CD25^(neg), CD27⁺,CD56^(pos/neg) , CD57^(neg), CD28^(hi), CD122⁺, CD127^(hi), PD-1^(neg),CD103^(neg), NKG2D^(int/lo), perforin^(lo/int), granzyme A^(int),granzyme B^(neg), Ki67^(neg), bcl-xL^(hi) and bcl-2^(hi).

PBMC were separated from fresh peripheral blood by density gradientcentrifugation and resuspended in PBS. Surface labeling was performedwith antibodies to CD4, CD16, TCRγδ, Vα24, CD3, CD8α, CD95, CD62L, CD161and other antibodies as indicated for 20 minutes on ice. After washingin cold PBS, surface-labeled samples were analyzed on a BD LSR-2 flowcytometer. Samples for intracellular staining were fixed in BD Cytofixbefore washing, permeabilization and labeling in BD Perm/wash buffer,then analyzed as above.

CMhi and EMhi subsets were identified as CD62L⁺/CD161^(hi) or CD62L⁻/161^(hi) events, respectively, in the CD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD3⁺/CD8⁺and CD95⁺ populations.

The results in FIGS. 2a-c ) demonstrate the unique phenotype of CMhi andEMhi subsets of CD8⁺ TCRαβ⁺ T cells. The phenotype is consistent with amemory population and is similar to murine memory stem cells asdescribed by Zhang et al (Nat Med, 2005). The phenotype is differentfrom that expected for other characterized lymphoid populations, such asNK cells and invariant NKT cells. CMhi and EMhi have a similarphenotype, but can be immunophenotypically differentiated by theexpression of CD62L. Only the phenotype of EMhi is shown for clarity inFIGS. 2a ) and b). FIG. 2c ) is shown to illustrate the higherexpression of bcl-2 and bcl-xl and lower expression of Ki67 in CMhi andEMhi, compared to CMlo and EMlo, from all donors tested.

Example 3 CMhi and EMhi Have a Surface Phenotype That SuggestsDerivation from the Distal Pole of an Asymmetrically Dividing CD8⁺ TCell or Uropod of a Polarized CD8⁺ T Cell

PBMC were separated from fresh peripheral blood by density gradientcentrifugation, washed in efflux buffer and resuspended at 1×10⁶/ml inice cold efflux buffer with 5 μg/ml rhodamine 123. PBMC were incubatedfor 30 minutes on ice before washing three times in ice cold effluxbuffer and resuspending in pre-warmed efflux buffer for 30 minutes at37° C. At 30 minutes, PBMC were washed once in ice cold PBS/0.2% BSA(FACS buffer) and labeled with antibodies to CD3, CD4, CD8, CD45RA,CD45RO, CD62L, CD16 and either CD11a, CD43, CD44, CD46, CD148 or CD162for 20 minutes on ice. After washing in ice cold FACS buffer, sampleswere analyzed on a BD FacsARIA flow cytometry. CM and EM subsets wereidentified as CD62L⁺ or CD62L⁻ events, respectively, in theCD4⁻/CD16⁻/CD3⁺/CD8⁺/CD45RA^(int/neg)/CD45RO⁺ population. Establishmentof rapid efflux was performed as described in Example 1.

FIG. 3 indicates that CMhi have a phenotype consistent with derivationfrom the ‘memory’ distal pole of a dividing CD8⁺ T cell or uropod of apolarized CD8+ T cell. The phenotype of CMhi is similar to the phenotypeof EMhi—only FACS profiles gated on CM CD8⁺ T cells are shown forclarity. The MFIs of CMhi and CMlo populations are shown in red. AfterTCR signaling in the presence of appropriate costimulation and/oradhesion molecules, an immune synapse forms between the APC and T cell.CD8 and CD11 a (amongst other cell surface proteins) actively localizeto the immune synapse (“Proximal markers”) and CD43, CD44, CD46, CD148and CD162 are excluded from the synapse and form the distal pole complex(“Distal markers”). A similar structure to the distal pole complex, theuropod, is also formed on stimulation of some T cells with chemokinesand has a similar pattern of expression of cell surface markers. It isunknown whether these surface molecules remain localized within oroutside the immune synapse or uropod until or beyond the first celldivision; however the phenotype of CMhi and EMhi, while variable, couldbe consistent with cell populations that are derived from the distalpole of a dividing memory cell or the uropod.

Example 4 CMhi and EMhi Express Higher Levels of MDR-1 mRNA Than TheirNon-effluxing Counterparts and Actively Efflux the FluorescentChemotherapy Drug, Daunorubicin

In FIG. 4(a), CMhi and EMhi and their non-effluxing IL-18Rα^(lo/neg)counterparts (CMlo and EMlo) were isolated using negative immunomagneticselection of CD8⁺ T cells with biotinylated antibodies to non-CD8⁺ Tcells, followed by surface labeling with fluorochrome-conjugatedstreptavidin to identify non-CD8⁺ T cells, CD95, CD62L and IL-18Rα andsorting on a BD FacsARIA flow sorter. CM and EM CD8⁺ T cells weredefined as streptavidin⁻/CD95⁺/CD62L⁺ or streptavidin⁻/CD95⁺/CD62L⁻,respectively. CMhi and EMhi were defined by high expression of IL-18Rα.Expression of mdr 1 in isolated CMhi, CMlo, EMhi and EMlo subsets wasdetermined by quantitative polymerase chain reaction, using thefollowing primers and probes: MDR1 forward—GGA AGC CAA TGC CTA TGA CTTTA; MDR1 reverse—GAA CCA CTG CTT CGC TTT CTG; MDR1 probe—6FAM-TGA AACTGC CTC ATA AAT TTG ACA CCC TGG-TAMRA. Results shown are normalized toGAPDH expression. The mean/SE from 3 normal donors are shown.

In FIG. 4(b), PBMC were resuspended at 1×10⁶/ml in efflux buffer andloaded with the fluorescent chemotherapy drug, daunorubicin, at 2.5 μMfor 20 minutes at 37° C., before washing three times and effluxing withor without vinblastine 25 μM for 1 hour at 37° C. PBMC were then washedonce in ice cold PBS/0.2% BSA (FACS buffer) and labeled with antibodiesto CD16, CD3, CD8α, CD95, CD62L and CD161 or IL-18Rα for 20 minutes onice. After washing in ice cold FACS buffer, samples were analyzed on aBD LSR-2 flow cytometer.

The data indicate that CMhi and EMhi express high levels of mRNA forMDR-1, the ATP-binding cassette (ABC) co-transporter responsible forspecific and active efflux of Rh123 and daunorubicin. The capacity ofCMhi and EMhi (defined by either high IL-18Rα or CD161 expression) toactively and specifically efflux a chemotherapy drug is shown by theinhibition of efflux in the presence of a competitive agonist for MDR-1protein, vinblastine. Inhibition was also achieved with two other MDR-1channel blockers, PK11195 and cyclosporine A (data not shown).

Example 5 CMhi and EMhi are Resistant to Daunorubicin-Induced ApoptosisIn Vitro

CMhi and EMhi and their non-effluxing IL-18Rα^(lo/neg) counterparts(CMlo and EMlo) were isolated using negative immunomagnetic selection ofCD8⁺ T cells with biotinylated antibodies to non-CD8⁺ T cells, followedby surface labeling with fluorochrome-conjugated streptavidin toidentify non-CD8⁺ T cells, CD95, CD62L and IL-18Rα and sorting on a BDFacsARIA flow sorter. CM and EM CD8⁺ T cells were defined asstreptavidin⁻/CD95⁺/CD62L⁺ or streptavidin⁻/CD95⁺/CD62L⁻, respectively.CMhi and EMhi were defined by high expression of IL-18Rα. Effluxing andnon-effluxing CM and EM subsets were cultured for 44 hours in thepresence or absence of daunorubicin (an anthracycline chemotherapeuticagent, effluxed by the ABC-B1 cotransporter, MDR-1), with or withoutMDR-1 blockade with the peripheral benzodiazepine receptor antagonist,PK11195. Cultures were harvested, washed twice with cold PBS and stainedwith Annexin V and DAPI before analysis.

The results in FIG. 5 indicate that CMhi and EMhi subsets are resistantto apoptosis induced by culture with daunorubicin at pharmacologicalconcentrations (0.1 uM). Chemoresistance is mediated by efflux pumps asinhibition of MDR-1 with PK11195 results in increased cell death.Culture with PK11195 alone does not impair viability.

Example 6 CMhi and EMhi Divide in Response to the Homeostatic Cytokines,IL-7 and IL-15, and Have High Viability After Culture in the Absence ofSupplementary Cytokines

In FIGS. 6(a-c), PBMC were separated from fresh peripheral blood bydensity gradient centrifugation. CD8⁺ T cells were positively selectedusing CD8 Microbeads (Miltenyi) and resuspended at 1×10⁶/ml in ice coldefflux buffer with 10 μg/ml Rh123. CD8⁺ T cells were incubated for 30minutes on ice before washing three times in ice cold efflux buffer andresuspending in pre-warmed efflux buffer for 30 minutes at 37° C.Vinblastine was added to control samples to establish the presence ofefflux). CD8⁺ T cells were then washed once in ice cold PBS/0.2% BSA(FACS buffer) and labeled with fluorochrome-conjugated antibodies toCD4, CD16, TCRγδ, Vα24, CD8α, CD95, CD62L and CD161. CMhi and EMhisubsets were identified as CD62L⁺/Rh123^(lo)/CD161^(hi) orCD62L⁻/Rh123^(lo)/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺ population. CMlo and EMlo subsets wereidentified as CD62L⁺/Rh123^(hi)/CD161^(int/neg) orCD62L⁻/Rh123^(hi)/CD161^(int/neg) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺ population. The gating strategy isshown in FIG. 6a ). Subsets were isolated using a BD FacsARIA flowsorter and proliferation in response to IL-7 was determined by³H-thymidine uptake or CF SE dilution assays. Proliferation in responseto IL-15 was determined by CFSE dilution assay. The ³H-thymidineproliferation assay was performed by culturing for 5 days in CTL mediumsupplemented with IL-7 then pulsing overnight with ³H-thymidine beforeharvesting and counting. The CFSE-dilution assay was performed byloading the cells with CFSE and culturing for 10 days, before viabilitylabeling with DAPI and analysis on a BD LSR2 flow cytometer.

In FIG. 6(d), PBMC from non-lymphopenic healthy donors (n=8) orlymphopenic acute myeloid leukemia patients (n=6) at the nadir (day 11to day 22) of induction therapy were analyzed for Ki67 expression onmemory subsets as described in Example 2. The fold change in the percentKi67 expression of CD8⁺ T cell subsets between non-lymphopenic healthydonors and lymphopenic patients is depicted.

The results indicate that the CMhi and EMhi subsets have the capacity toenter the cell cycle and undergo division in response to IL-7 (FIG. 6b )and IL-15 (FIG. 6c ) stimulation. CMhi and EMhi are also recruited moreeffectively into the cell cycle than CMlo and EMlo in lymphopenicchemotherapy patients (FIG. 6d ), suggesting high sensitivity tolymphopenia-induced IL-7 and IL-15-mediated proliferation. In addition,CMhi and EMhi maintain higher viability in culture in the absence ofsupplementary cytokines than their non-effluxing counterparts (FIG. 6b). IL-7 and IL-15 are critical for the maintenance of long term memoryand survival of memory T cells and the CMhi and EMhi subsets aresensitive to signaling by IL-7 and IL-15.

Example 7 CMhi and EMhi Show Reduced ³H-thymidine Uptake, Compared toTheir Non-effluxing Counterparts, in Response to Polyclonal TCRStimulation with OKT3, and Can be Rescued After Costimulation asIndicated in FIG. 7

CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMCwere separated from fresh peripheral blood by density gradientcentrifugation. CD8⁺ T cells were positively selected using CD8paramagnetic beads and resuspended at 1×10⁶/ml in ice cold efflux bufferwith 10 μg/ml Rh123. CD8⁺ T cells were incubated for 30 minutes on icebefore washing three times in ice cold efflux buffer and resuspending inpre-warmed efflux buffer, with or without vinblastine, for 30 minutes at37° C. At 30 minutes, CD8⁺ T cells were washed once in ice cold PBS/0.2%BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies toCD4, CD16, TCRγδ, Vα24, CD8α, CD95, CD62L and CD161. CMhi and EMhisubsets were identified as CD62L⁺/Rh123^(lo)/CD161^(hi) orCD62L⁻/Rh123^(lo)/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺ population. CMlo and EMlo subsets wereidentified as CD62L⁺/Rh123^(hi)/CD161^(int/neg) orCD62L⁻/Rh123^(hi)/CD161^(int/neg) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺ population. Subsets were isolatedusing a BD FacsARIA flow sorter and cultured in 96 well plates at10,000-30,000 per well in 200 μl CTL in the indicated conditions. OKT3was plate-bound by incubating at 1000 ng/ml in 100 μl PBS per well for 6hours at 4° C., then washing twice with 200 ul cold PBS before platingthe sorted subsets. Anti-CD28 was plate-bound (at 5 μg/ml) with OKT3, asabove. Cytokine concentrations were as follows: IL-7, 2 ng/ml; IL12, 10ng/ml; IL-15, 1 ng/ml, IL-18, 80 ng/ml; IL-23, 10 ng/ml. Culture withcytokines in the absence of cytokine costimulation resulted in minimalproliferation. Data for the proliferation of the CMhi subset with IL-12alone or OKT3/IL-12 is not available.

Example 8 CMhi and EMhi Have a Different Cytokine Secretion ProfileCompared to Their Non-effluxing Counterparts

CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMCwere separated from fresh peripheral blood by density gradientcentrifugation. CD8⁺ T cells were positively selected using CD8paramagnetic beads and resuspended at 1×10⁶/ml in ice cold efflux bufferwith 10 μg/ml Rh123. CD8⁺ T cells were incubated for 30 minutes on icebefore washing three times in ice cold efflux buffer and resuspending inpre-warmed efflux buffer, with or without vinblastine, for 30 minutes at37° C. At 30 minutes, CD8⁺ T cells were washed once in ice cold PBS/0.2%BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies toCD4, CD16, TCRγδ, Vα24, CD8α, CD95, CD62L and CD161. CMhi and EMhisubsets were identified as CD62L⁺/Rh123^(lo)/CD161^(hi) orCD62L⁻/Rh123^(lo)/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺ population. CMlo and EMlo subsets wereidentified as CD62L⁺/Rh123^(hi)/CD161^(int/neg) orCD62L⁻/Rh123^(hi)/CD161^(int/neg) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺ population. Subsets were isolatedusing a BD FacsARIA flow sorter and plated in 200 μl CTL medium at60,000 cells per well. Polyclonal stimulation was performed by culturingisolated subsets with either PMA (5 ng/ml)/ionomycin (1 μg/ml) orplate-bound OKT3/anti-CD28 (prepared as described in Example 7) for 20hours. Cytokine secretion was detected in culture supernatant using aLuminex Cytokine Array assay.

These experiments show that CMhi and EMhi secrete less IL-2, IL-4, IL-6,IL-8, IL-10, IFN-γ and MIP-1α and more IL-17 than their non-effluxingcounterparts in response to polyclonal stimulation.

Example 9 CMhi and EMhi Have Decreased Calcium Flux in Response toIonomycin, Compared to Their Non-effluxing Counterparts

PBMC were separated from fresh peripheral blood by density gradientcentrifugation at room temperature and incubated at 1×10⁷/ml in CTLmedium supplemented with Indo-1AM (Sigma) 10 μM and probenicid 4 mM for30 minutes at 37° C. The PBMC were washed once in CTL medium at 25° C.Surface labeling was performed in CTL medium with antibodies to CD4,CD16, TCRγδ, Vα24, CD8α, CD62L and CD161 for 10 minutes at roomtemperature. After washing in room temperature CTL medium,surface-labeled samples were warmed to 37° C. for 4 minutes before highspeed acquisition on a BD LSR-2 flow cytometer equipped with UV, violet,blue, green and red lasers. After 30 seconds acquisition, the sample wasremoved from the aspiration port, ionomycin was added to a finalconcentration of 5 μg/ml, the sample was returned and acquisition wascontinued at 20,000 events/second. CMhi and EMhi subsets were identifiedas CD62L⁺/CD161^(hi) or CD62L⁻/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺ populations. CMlo and EMlo subsetswere identified as CD62L⁺/CD161^(int/neg) or CD62L⁻/CD161^(int/neg)events, respectively, in the CD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺populations. Relative intracellular calcium concentration was measuredas the ratio of Indo-1AM fluorescence in the UV violet (405 nm):UV blue(505 nm) detectors and is plotted as the mean ratio versus time(seconds) for appropriately gated subsets.

This experiment demonstrates that CMhi and EMhi subsets have a differentcapacity to flux calcium in response to the calcium ionophore,ionomycin, compared to their non-effluxing counterparts. Calcium flux isa proximal signaling event downstream from antigen-specific TCRligation.

Example 10 CMhi and EMhi Subsets Comprise Polyclonal TCR Repertoires byMolecular Spectratyping, as Described in Example 10

CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMCwere separated from fresh peripheral blood by density gradientcentrifugation. CD8⁺ T cells were positively selected using CD8paramagnetic beads and resuspended at 1×10⁶/ml in ice cold efflux bufferwith 10 μg/ml Rh123. CD8⁺ T cells were incubated for 30 minutes on icebefore washing three times in ice cold efflux buffer and resuspending inpre-warmed efflux buffer, with or without vinblastine, for 30 minutes at37° C. At 30 minutes, CD8⁺ T cells were washed once in ice cold PBS/0.2%BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies toCD4, CD16, TCRγδ, Vα24, CD8α, CD95, CD62L and CD161. CMhi and EMhisubsets were identified as CD62L⁺/Rh123^(lo)/CD161^(hi) orCD62L⁻/Rh123^(lo)/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺ population. CMlo and EMlo subsets wereidentified as CD62L⁺/Rh123^(hi)/CD161^(int/neg) orCD62L⁻/Rh123^(hi)/CD161^(int/neg) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺ population. Naïve CD8⁺ T cells wereidentified as CD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁻/CD62L⁺ events. Subsetswere isolated using a BD FacsARIA flow sorter.

Molecular Vβ spectratyping was performed on isolated subsets and naïveCD8⁺ T cells by multiplex RT-PCR and Genescan analysis of TCR Vβfragments.

These experiments show polyclonal TCR Vβ usage in the effluxing CMhi andEMhi subsets, demonstrating that the CD8⁺ T cells within the CMhi andEMhi subsets express diverse TCR that are potentially specific for abroad range of antigens.

Example 11 Viral Antigen Tetramer-Positive Cells Can be Identifiedwithin CMhi and EMhi Subsets and CMV-, EBV- and Influenza-Specific CTLResponses Can be Generated from Sorted CMhi and EMhi Subsets, asDescribed in Example 11

PBMC were separated from fresh peripheral blood by density gradientcentrifugation and resuspended in PBS. Surface labeling was performedwith antibodies to CD4, CD16, TCRγδ, CD8α, CD95, CD62L, CD161 and anAPC-labeled HLA-A*0201; NLV peptide tetramer to allow identification ofCD8⁺ T cells specific for the NLV peptide from the pp65 antigen of CMV.After washing in cold PBS, surface-labeled samples were analyzed on a BDLSR-2 flow cytometer. CMhi and EMhi subsets were identified asCD62L⁺/CD161^(hi) or CD62L⁻/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/CD8⁺/CD95⁺ populations. Data is shown in FIG. 11a ).

To demonstrate that rare tetramer-positive events seen in the CMhi andEMhi subsets ex vivo were viral antigen-specific CTL, we expandedantigen-specific CTL in vitro from isolated CMhi and EMhi subsets byculture with autologous activated peptide-pulsed monocyte-derived DC(MoDC). CMhi, CMlo, EMhi and EMlo were isolated as described in Example6. MoDC were generated by culture of CD14⁺ monocytes, isolated usingCD14-specific paramagnetic beads, with GM-CSF (800 U/ml) and IL-4 (1000U/ml) for 5 days. Mature MoDC were generated by culture to day 7 withadditional GM-CSF (800 U/ml)/IL-4 (1000 U/ml) and IL-1β(2 ng/ml), IL-6(1000 U/ml), PGE₂ (1000 ng/ml) and TNFα (10 ng/ml). Activated MoDC werepulsed in RPMI1640 (Gibco) for 2 hours at room temperature with 1 μg/mlHLA-A*0201-restricted NLVPMVATV, GLCTLVAML or GILGFVFTL peptides,derived from CMV, EBV or influenza, respectively. MoDC were washed 3times in RPMI1640 and irradiated (3500 cGy) before use.

CMhi, CMlo, EMhi and EMlo subsets were plated in 96 well plates withirradiated, activated, peptide-pulsed MoDC at a T:DC ratio of 4:1 in 200ml CTL medium supplemented with IL-2 (10 U/ml), IL7 (1 ng/ml) and IL-15(100 pg/ml). Cytokine and half medium exchanges were performed on days 4and 7 and analysis by CD8, DAPI and tetramer staining was performed onday 10. Data is shown in FIG. 11b ).

This experiment demonstrates that rare virus-specific tetramer-positiveCD8⁺ T cells can be identified within rapidly effluxing CMhi and EMhipopulations ex vivo and that rare viral antigen-specific CD8⁺ T cellscan be identified after in vitro stimulation of isolated effluxing CMhiand EMhi subsets. Despite the fact that effluxing CMhi and EMhi arerefractory to stimulation with OKT3 (Example 7), proliferation can berescued by culturing with cytokines. The use of activated MoDC andcytokine supplementation in Example 11 allows expansion ofantigen-specific CTL from effluxing subsets in vitro.

Example 12 CMhi and EMhi Subsets Have Unique and Distinct GeneExpression Profiles

CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMCwere separated from fresh peripheral blood by density gradientcentrifugation. CD8⁺ T cells were positively selected using CD8-specificparamagnetic beads and resuspended at 1×10⁶/ml in ice cold efflux bufferwith 10 μg/ml Rh123. CD8⁺ T cells were incubated for 30 minutes on icebefore washing three times in ice cold efflux buffer and resuspending inpre-warmed efflux buffer, with or without vinblastine, for 30 minutes at37° C. At 30 minutes, CD8⁺ T cells were washed once in ice cold PBS/0.2%BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies toCD4, CD16, TCRγδ, Vα24, CD8α, CD95, CD62L and CD161. CMhi and EMhisubsets were identified as CD62L⁺/Rh123^(lo)/CD161^(hi) orCD62L⁻/Rh123^(lo)/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺ population. CMlo and EMlo subsets wereidentified as CD62L⁺/Rh123^(hi)/CD161^(int/neg) orCD62L⁻/Rh123^(hi)/CD161^(int/neg) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺ population. Subsets were isolatedusing a BD FacsARIA flow sorter.

cRNA was generated from isolated subsets and gene expression arraystudies were performed, using the Illumina HumanWG-6 expression beadchiparray. The data are displayed on a Principal Components Plot toillustrate the gene expression relationships of CMhi and EMhi inrelation to non-effluxing CD8⁺ T cell subsets. The data show thatrapidly effluxing (CMhi and EMhi) subsets have gene expression profilesthat are distinct from those of naïve or non-effluxing memory (CMlo andEMlo) CD8⁺ T cells. In addition, the separation of CMhi and EMhiclusters suggests that, despite their similar phenotype, CD62L⁺ (CMhi)and CD62L⁻ (CMlo) effluxing CD8⁺ T cells have different gene expressionprofiles.

Example 13 CMhi and EMhi are Rare in Cord Blood, Peak in Early AdultLife and are Found at Decreasing Frequency with Advancing Age

PBMC were separated from fresh peripheral blood or cord blood by densitygradient centrifugation. CD8⁺ T cells were positively selected using CD8Microbeads (Miltenyi) and resuspended at 1×10⁶/ml in ice cold effluxbuffer with 10 μg/ml Rh123. CD8⁺ T cells were incubated for 30 minuteson ice before washing three times in ice cold efflux buffer andresuspending in pre-warmed efflux buffer for 30 minutes at 37° C.Vinblastine was added to control samples to establish the presence ofefflux). CD8⁺ T cells were then washed once in ice cold PBS/0.2% BSA(FACS buffer) and labeled with fluorochrome-conjugated antibodies toCD4, CD16, TCRγδ, Vα24, CD8α, CD95, CD62L and CD161. CMhi and EMhisubsets were identified as CD62L⁺/Rh123^(lo)/CD161^(hi) orCD62L⁻/Rh123^(lo)/CD161^(hi) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺ population. CMlo and EMlo subsets wereidentified as CD62L±/Rh123hi/CD161^(int/neg) orCD62L⁻/Rh123^(hi)/CD161^(int/neg) events, respectively, in theCD4⁻/CD16⁻/TCRγδ⁻/Vα24⁻/CD8⁺/CD95⁺ population. The gating strategy isshown in FIG. 6a ). Samples were assayed using a BD FacsARIA flowcytometer. The frequency of CMhi and EMhi phenotype cells as apercentage of CD8⁺ T cells is shown in cord blood compared to adultperipheral blood in FIG. 13a ). The percentage of effluxing (top) andnon-effluxing (bottom) cells in the parental CM and EM compartments areshown in FIG. 13b ). Each point represents a single healthy donor.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A composition comprising an isolatedpopulation of long-lived human memory CD8⁺ CD161^(hi) IL-18Rα^(hi) Tcells, wherein: the long-lived memory CD8+ CD161^(hi) IL-18Rα^(hi) Tcells make up at least 30% of the total CD8+ T cells in the composition;and the long-lived memory CD8+ CD161^(hi) IL-18Rα^(hi) T cells compriseCD95^(hi) memory cells, wherein the CD95^(hi) memory cells are capableof proliferating in response to IL-7 or IL-15 and comprise a populationof cells comprising an engineered immunoreceptor.
 2. The composition ofclaim 1, wherein the population of long-lived memory CD8+CD161^(hi)IL-18Rα^(hi) T cells comprises high CD28 surface expressionand high MDR-1 mRNA levels as compared to a CD8+ T cell population withlow surface expression of IL-18Rα.
 3. The composition of claim 1,wherein the long-lived memory CD8+ CD161^(hi) IL-18Rα^(hi) T cells areCD127⁺, CD25⁻, bcl−2^(hi), perforin^(neg/low), granzyme A^(int),granzyme B^(int/neg) and NKG2D^(int).
 4. The composition of claim 1,wherein the long-lived memory CD8+ CD161^(hi) IL-18Rα^(hi) T cellpopulation has increased expression of CD43, CD44, CD46, CD148, andCD162 as compared to a CD8+ T cell population with low surfaceexpression of IL-18Rα.
 5. The composition of claim 1, wherein thelong-lived memory CD8+ CD161^(hi) IL-18Rα^(hi) T cell population lacksexpression of CD57, CD103, and CD69 as compared to a CD8+ T cellpopulation with low surface expression of IL-18Rα.
 6. The composition ofclaim 1, wherein the long-lived memory CD8+ CD161^(hi) IL-18Rα^(hi) Tcell population increased expression of CD122 as compared to a CD8+ Tcell population with low surface expression of IL-18Rα.
 7. Thecomposition of claim 1, wherein the CD95^(hi) memory cells compriseCD62L⁺, CD45RA^(int/neg), CD45RO^(int/hi) central memory cells.
 8. Thecomposition of claim 7, wherein the CD95^(hi) memory cells furthercomprise CD62L⁻, CD45RA^(int/neg), CD45RO^(int/hi) central memory cells.9. The composition of claim 1, wherein the CD95^(hi) memory cellscomprise CD62L⁻, CD45RA^(int/neg), CD45RO^(int/hi) central memory cells.10. The composition of claim 1, wherein the engineered immunoreceptor isspecific for a tumor-associated antigen.
 11. The composition of claim 1,wherein the engineered immunoreceptor is an antigen-specific T cellreceptor.
 12. The composition of claim 1, wherein the long-lived memoryCD8+ CD161^(hi) IL-18Rα^(hi) T cells are at least 40% of the total CD8+T cells in the composition.
 13. The composition of claim 1, wherein thelong-lived memory CD8+ CD161^(hi) IL-18Rα^(hi) T cells are at least 50%of the total CD8+ T cells in the composition.
 14. The composition ofclaim 1, wherein the long-lived memory CD⁸+ CD161^(hi) IL-18Rα^(hi) Tcells are at least 80% of the total CD8+ T cells in the composition. 15.The composition of claim 1, wherein the long-lived memory CD8+CD161^(hi)IL-18Rα^(hi) T cells have enhanced proliferation in responseto a cytokine selected from the group consisting of IL-12, Il-18, IL-23,or combinations thereof, as compared to a CD8+T cell population with lowsurface expression of IL-18Rα.